Gastrin-releasing peptide (GRP) is widely distributed in
the central nervous system, and is involved in a variety of physiological
processes and behaviors, including the circadian rhythm, food intake,
anxiety/fear responses, and male sexual function. Recently, the spinal GRP/GRP-receptor system
has been identified as an itch-specific mediator in the somatosensory
system. We demonstrated that the expression of GRP was predominant in the small-sized neurons of the
primary afferents, and GRP-immunoreactive axons terminated the superficial
layers of the spinal dorsal horn in rats.
Furthermore, ultrastructure of GRP-immunoreactive axon terminals was
analyzed by various kinds of electron microscopy. The analysis of high-voltage electron
microscopy showed that GRP-immunoreactive axon terminals formed a series of the
varicosities. The analysis of transmission
electron microscopy showed the GRP-immunoreactive presynaptic terminals
contained many clear microvesicles and large dense-cored vesicles. Serial block-face scanning electron microscopy
(SBF-SEM) that combines SEM and ultramicrotome makes serial
ultrathin sections automatically. Until now was technically impossible,
but the reconstruction of a three-dimensional ultrastructure has become possible owing to this electron microscopy.We have developed a technology that applies immunohistochemistry to label the target molecule in this microscope. SBF-SEM analysis showed that not one but some
dendritic spines contacted a single varicosity containing GRP-immunoreactive
dens-cored vesicles. Thus,
by combination of several kind of electron
microscopy, visualization of a three-dimensional
ultrastructure of neuropeptide in the axon terminals has
become possible.